Development of Highly Sensitive Excitation Ratiometric Indicators of Cellular Phosphorylation

Biotechnology Medical Research Tools

Keywords biosensor, enzymatic regulation, phosphorylation, in vivo assays, enzyme activity reporter, kinase sensor, endogenous kinase activity, drug screening


Protein phosphorylation is one of the most common forms of post-translational modification and is involved in the regulation of key signaling pathways in the cell. Dysfunctional phosphorylation plays a key role in various diseases, especially cancer and neurodegenerative disorders. Protein kinases have been the focus of intense recent interest by the pharmaceutical industry. Indeed, most new cancer drugs approved by the FDA in the last several years target kinases, and there are hundreds of new kinase inhibitors under development. To this end, it is very important to have quantitative methods for measuring changes in kinase activities. In vitro kinase activity assays take the target molecules out of cellular contexts. Fluorescent protein-based kinase biosensors have enabled the real-time monitoring of kinase activities within the native context of living cells, yet most commonly used biosensors exhibit poor sensitivity (e.g., dynamic range) for imaging physiological signaling activities in situ .

Technology description

Researchers at UC San Diego have developed a new class of excitation ratiometric kinase activity biosensors that offer the highest reported dynamic range and enable the detection of subtle changes in signaling activity that could not be reliably detected previously. These high-performance molecular tools permit the robust, sensitive detection of subtle activity changes in vivo and thus further expand the frontiers of “native biochemistry”. The invented reporters are based on phosphorylation-dependent changes in Green Fluorescent Protein (GFP) emission intensity upon excitation with 480 nm versus 400 nm light. For example, in the 1st generation excitation ratiometric A-kinase activity reporter (ExRai-AKAR1), PKA activity elevations induce a ~25% increase in 480 nm-stimulated fluorescence emission and a ~50% decrease in 400 nm-stimulated fluorescence emission, which translates into a ~150% increase in the 480/400 excitation ratio. A more optimized ExRai-AKAR2 variant produces up to a 1000% increase in the 480/400 excitation ratio in response to PKA activation .


The invented indicators could be commercialized as powerful biotechnological tools for in situ protein kinase activity assay. The improved versions could be used in high-throughput assays for drug screening or for diagnostic analysis.


The invented reporters are unique for being the first excitation ratiometric kinase sensors ever described in the literature. A key advantage of the reporter design over the existing state of the art is that the excitation ratiometric behavior produces dramatically higher fluorescence responses and vastly improved signal-to-noise ratios compared with existing genetically encoded kinase activity reporters, thereby permitting much more sensitive detection of minute, physiologically relevant changes in kinase activity that were not detectable previously. Furthermore, it offers

  • Real-time monitoring offering instant readout
  • Live-cell imaging with no cell lysis required
  • Detection of endogenous kinase activity
  • Ratiometric readout for reliable quantification

State of development

The invention has yielded working prototype indicators for several kinases (currently up to four) and we have generated optimized versions of some of these through random and targeted mutation.

Intellectual property info

This technology is patent pending and available for licensing and/or research sponsorship.

Patent status

Patent Pending

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